Human erythrocyte membranes exhibit a cooperative calmodulin-dependent Ca²⁺-ATPase of high calcium sensitivity

It is thought that the ionized Ca²⁺ concentration in the cytosol of healthy erythrocytes is in the range 0.01-0.1 μM (ref. 1) and that this low concentration is maintained by an ATP-driven calmodulin-dependent Ca ²⁺ pump in the plasma membrane^2,3. The Ca ²⁺-stimulated ATPase which is the enzymatic expression of this pump varies in its calcium sensitivity between different preparations of erythrocyte ghosts, with activation generally occurring in a concentration range between ∼1 and 10 μM Ca²⁺ (refs 2-5). This is a higher range of Ca²⁺ concentrations than might be anticipated for activation of a pump that sustains intracellular concentrations of Ca²⁺ below 0.1 μM, and recent reports have suggested activation in some membrane preparations at Ca²⁺ concentrations in the range 0.1-1.0 μM (refs 6, 7). We report here a simple method for preparing human erythrocyte membranes in 2.5 mM HEPES/1 mM EGTA at pH 7.0 (see Fig. 1 legend) in which the activation of the Ca²⁺-ATPase by Ca²⁺ and intracellular concentrations of calmodulin is highly cooperative and is complete by ∼1 μM Ca²⁺. Unlike other available erythrocyte membrane preparations, the pattern of activation by Ca²⁺ and calmodulin is not complicated by partial resealing of the ghosts during and after isolation. We suggest that this cooperative activation of the Ca²⁺ pump may explain how healthy erythrocytes maintain their normal cytosol Ca²⁺ concentration at a threshold value at or below ∼0.1 μM. We also note that several other calmodulin-dependent enzymes display similar cooperative activation kinetics.